In hybridization reactions a labelled oligo- or polynucleotide, i.e. the probe is allowed to base-pair with the nucleic acid target. Various hybridization methods have been used for the detection of nucleic acids. In direct hybridization methods the specimen is either in solution or fixed to a solid carrier. The nucleic acid which is to be identified is demonstrated using one labelled probe.
In U.S. Pat. No. 4,486,539 a sandwich hybridization method has been described. In this method two separate probes are used, one being a detector probe labelled and used for detection and the other being a capturing probe immobilized to a solid carrier for the separation of the target nucleic acid from the reaction mixture.
The method of hybridization in solution is described in British Patent Publication No. 2 169 403. Two different probes both being in the same solution phase are used in this method. The detector probe is labelled with a detectable label and to the capturing probe a moiety having affinity for another component is attached. After the hybridization the hybrid formed between the capturing probe, target nucleic acid and the detector probe, may be separated from the hybridization solution by the aid of the other moiety of the affinity pair.
The enzyme catalyzed polymerization of DNA where the nucleotide sequence of a previously existing nucleic acid strand, i.e. the template is accurately copied into its complementary strand, is well-known in the art and has been described e.g. in Kornberg, DNA replication, W. H. Freeman & Co, San Francisco, pp. 221-225 and 670-679, 1980 and Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, p. 122, 1982. This biological multiplication is used in hybridization assays in which the microbe to be detected is cultivated and hence its DNA enriched prior to the test and is described e.g. in Woo, Methods Enzymol. 68, p. 389, 1979 and in U.S. Pat. No. 4,358,535. Specific DNA sequences can also be amplified within living cells e.g. by the use of suitable drugs as described by Clewell and Helinski in J. Bacteriol. 110, p. 1135, 1972 and in European Patent Application No. 55 742. A more specific DNA-enrichment is described in in the European Patent Application No. 175 689 in which the target is linked to a plasmid replicon and introduced into a suitable cell. Yet another method is described in the European Patent Application No. 201 184, in which the primer dependence of DNA synthesis is utilized to create an in vitro reaction for the amplification of the target DNA. In the European Patent Application No. 200 362 a method for detecting amplified genes is suggested.